The properties of a polymer are of great importance in any application. For biomedical polymers, the most important single property is probably biocompatibility, which refers to the interactions of living body tissues, compounds and fluids including blood with any implanted or contacting polymeric material. Each system of polymer-body tissue interactions must be studied individually in terms of polymer stability, general tissue-fluid interactions and blood compatibility.
Polyurethane block copolymers have been proposed for use in blood-contacting applications because of their generally excellent physical properties and relatively good blood compatibility. Lelah and Cooper, Polyurethanes in Medicine, CRC Press, Boca Raton. Fla. (1986). It is desirable however, to further improved the blood compatibility of these materials to allow their use in such demanding applications as small-diameter vascular grafts, catheters, kidney dialyzers, cardiac assist devices and the artificial heart.
Thrombus formation on polyurethanes and other blood-contacting biomaterials can lead to occlusion of vascular grafts or catheters, and detachment (embolization) of these thrombi may result in tissue damage or strokes. Several approaches have been proposed for improving the blood compatibility of blood-contacting biomaterials. One method involves the preparation of a highly hydrophilic, mobile interfaces comprising materials that appear "bland" to blood components. Merrill et al., Am. Soc. Artif. Intern. Organs J., 6, 60 (1983). These "bland" materials may demonstrate a reduced tendency for protein absorption and platelet adhesion.
Another method involves the introduction of highly hydrophobic groups to the blood-contacting interface, either by using a highly hydrophobic polymer such as silicone rubber or by grafting long alkyl chains to a relatively hydrophilic material, such as a polyurethane block copolymer. By using long-chain alkyl grafting methods, Eberhart and coworkers [Munro, et al., Am. Soc. Artif. Intern. Organs J., 6, 65 (1983)] have improved the blood compatibility of polyurethane block copolymers. Similar approaches have resulted in the reduction of platelet and fibrinogen deposition in canine ex vivo blood-contacting studies.
A third method involves incorporating ionic character into the polymer. This approach may also result in a highly hydrophilic and mobile interface which has a low driving force for protein adsorption and cell adhesion. In fact, many researchers have used this approach in an attempt to provide a surface that will initiate the anti-coagulant action of heparin, a highly ionic mucopolysaccharide.
The electrical nature of a polymeric substrate is an important determinant of interfacial energetics. Many components of mammalian blood, including red blood cells, platelet surfaces, plasma proteins, and morula vascular endothelium, are negatively charged at physiological pH. Thus, ionic groups play an important, but not yet fully understood, role in blood-material interactions.
Sawyer and Pate [Born, Ann. NY Acad. Sci., 201, 4 (1972)] demonstrated that normal vascular endothelium is negatively charged and proposed that the natural blood vessel is thromboresistant due to repulsion between negative charges on the vessel wall and the blood components. Sawyer et al., Bull. NY Acad. Med., 48, 235 (1972) also examined a number of metals and demonstrated that relatively electronegative metals are less thrombogenic than others. One study concluded that polymers with a negative zeta potential, including an acrylic latex combined with a sulfonate detergent, carboxyl cellulose and fluorinated silicones are relatively blood compatible. (Rembaum et al., Polym. Prepr., 16, 191 (1975).
Negative charge by itself, however, is not sufficient to impart thromboresistance to a material. Glass, for example, is a well-known coagulant in spite of this relatively large negative zeta potential. In studies of the blood compatibility of silicone rubber, Musolf et al., NIH PB, 90, 666 (1969) found that carboxylation failed to improve the observed blood compatibility. Hageman Factor (an intrinsic blood clotting factor) has been shown to be activated by a variety of negatively charged surfaces. Nossel et al., Nature, 221, 74 (1969). Thus, the mechanism of action for a negatively-charged species such as heparin in coagulation inhibition is believed to be much more complex than merely the action of the negative charge.
It is well-known that plasma proteins are rapidly adsorbed when blood contacts an artificial surface, and it is believed that this protein layer influences the thrombogenicity of the surface. For example, the amount of platelet activation appears to be strongly mediate by the adsorbed protein layer. Park et al., J. Biomed. Mater. Res., 20, 589 (1986). The degree of platelet spreading which is promoted by the surface and the adsorbed protein layer is an important parameters for controlling the thrombogenicity of the surface and has been found to affect the relative amounts of proteins which adsorb at the interface. Weathersby et al., J. Bioeng., 1, 395 (1977); Baszkin et al., J. Biomed. Mater. Res., 14, 393 (1980) and Van Dulm et al., J. Coll. Int. Sci., 91, 248 (1983).
More direct investigations of surface charge effects on protein adsorption have been attempted, but conflicting results have been obtained. It has been found, for example, that ionic character in itself has relatively minor effects on protein adsorption when compared to other parameters. Schmitt et al., J. Coll. Int. Sci., 92, 25 (1983); Morrissey et al., J. Coll. Int. Sci., 56, 537 (1976) and Norde et al., J. Col. Int. Sci., 66, 257 (1978). Van Dulm et al., J. Coll. Int. Sci., 56, 557 (1976) reported that in one case albumin adsorption behavior differed markedly from other situations, and relatively slow initial adsorption rates were observed in the special case where both species were negative charged.
It has also been demonstrated that polymers with carboxylate functional groups interact with proteins in a different manner than those with sulfonate groups. Bernfeld, P., "Interaction of Polyanions with Blood Components", in The Amino Sugars; Balaz E. A. and Jeanloz, R. W. (eds) Academic Press, New York, 251-256 (1966) and Gelman et al., Biopolymers, 12, 541 (1973). Recent work by Fougnot et al., Biomaterials, 5, 89 (1984) has shown that the binding of sulphamide and/or sulfonate groups to a substrate products surfaces with relatively high affinities for albumin, thrombin and antithrombin. These substrates were shown to be relatively blood-compatible when compared to other surfaces.
However, the effect of ionic character on protein adsorption and subsequent thrombogenesis is still quite controversial. Muramatsu et al., J. Biomed. Mater. Res., 17, 959 (1983) studied protein adsorption using static adsorption methods to determine adsorption isotherms on artificial red blood cells surfaces. It was determined that the surface negative charge of the surfaces, as evidenced by the relative number of sulfonic acid groups present, strongly affected the composition, molecular orientation, and/or configuration of adsorbing plasma components. Fibrinogen and gammaglobulin adsorption were particularly affected by surface charge.
The mechanism of heparin action has also been considered in anticoagulation research. Heparin is a naturally-occurring mucopolysaccharide anticoagulant. Its molecular weight ranges from below 10,000 to above 20,000, with the higher molecular weight fraction generally showing a higher level of anti-coagulant activity. Ebert et al., "The Anticoagulant Activity of Derivatized and Immobilized Heparins", in Biomaterials: Interfacial Phenomena and Applications, Cooper, S. L. and Peppas, N. A. (eds.), ACS Adv. in Chem. Services, 199, 161 (1982). The mechanisms by which heparin exerts its anticoagulant function are not well-defined. Ebert et al., id.; Jozefowicz et al., Pure and Appl. Chem., 56, 1335 (1984) and Olsson et al., Ann. NY Acad. Sci., 416, 525 (1984).
The most potent plasma inhibitor of the coagulation process is antithrombin III (AT-III), which forms inactive stable complexes with serine-proteases including clotting factors IIa, IXa, Xa, IXa and kallikrein. These reactions are believed to be subject to catalysis by heparin and heparin analogs which might be present in some subendothelial or endothelial tissue. The generally accepted scheme is that heparin binds to AT-III and greatly potentiates thrombin binding to AT-III binding sites in the heparin AT-III complex. The complex not only binds to thrombin, but also binds to every active serine protease in the intrinsic coagulation pathway.
Lindsay et al., Trans. Am. Soc. Artif. Inter. Organs, 22, 292 (1976) cited a number of conflicting reports on the action of heparin on platelets and concluded that variations in experimental techniques probably account for the many contradictory findings. Their own in vitro platelet retention study indicated that heparin had two antagonistic effects in platelet-foreign surface interactions. First, heparin in the blood was found to act directly on platelets to increase their retention. Second, the reduction of platelet adhesion to surfaces to which heparin was ionically attached led to the conclusion that heparin acted on the foreign surface, probably by competing for cationic sites. This suggests that heparinized surfaces may be passivated, but the heparin may not be performing its normal biological function.
It was found by Gott et al., Trans. Am. Soc. Artif. Inter. Organs, 10, 213 (1964) that heparin ionically bound to a polymer surface tends to result in a decreased tendency for the surface to promote coagulation. Since that time, the covalent and ionic binding of heparin and other anticoagulants have been the subject of numerous studies. Jozefowicz et al., id. and Olsson et al., id. review these studies. While ionically bound heparin has demonstrated antithrombogenic characteristics, materials heparinized in this manner have been effective only while they release the ionically bound heparin into the bloodstream. This mechanism is not satisfactory for long-term implantation. Van der Lei et al., Trans. Am. Soc. Artif. Intern. Organs, 31, 107 (1985) found that ionically-bound heparin does not increase patency in small diameter polyurethane vascular grafts.
Covalently-bonded heparin surfaces have been developed; and in most, but not all cases [Hashimoto, K., Tokohu J. Exp. Med., 81, 93 (1963)], an imporvement in antithrombogenicity of the derivatized surfaces with respect to the untreated surfaces has been shown. In agreement with the foregoing discussion, it has been noted that the surfaces to which heparin is covalently bound tend to activate platelets when exposed to blood or a platelet suspension (Jozefowicz et al., id.). A surface modification of a BIOMER polyurethane vascular graft involving covalently-bound heparin did not result in decreased platelet or fibrinogen deposition in two canine ex vivo experiments. Lelah M. D., Ph.D. Dissertation, Univ. of Wisconsin-Madison (1984).
Recent results by Sharma et al., ACS Div. Polymer. Mat. Sci. Eng. Prep. 53, 423 (1985) indicated that some heparinization methods for polyurethanes resulted in sharply increased fibrinogen adsorption from in vitro competitive adsorption experiments, and also provided a dramatic decrease in platelet adhesion from platelet-rich plasma. These investigators attributed their findings to specific interactions between the platelets and heparin, and did not consider such factors as fibrinogen conformational changes which could result upon adsorption to the different surface.
In any case, the use of covalent heparin binding to achieve an antithrombogenic surface may be limited use, as exposure to blood is expected to eventually degrade the bound heparin under the action of heparinases.
The development of "heparinoid" materials has generally involved the synthesis of polyelectrolytes with sulfonate and/or carboxylate functionality. Arge, E., ACTA Med. Scand., 155, 496 (1956) and Walker et al., Biochem. Biophys. Res. Comm., 83, 1339 (1978) have hypothesized that the mechanism of heparin action is based on the action of the sulfate and aminosulfate groups on the heparin molecule. Hashimoto, id., also asserted that sulfate or sulfonate groups might simulate the action of a heparinized surface and prevent thrombus formation. Conflicting results were reported by Olsson et al., id., who prepared surfaces of sulfated polysaccharrides and found them to be equally "platelet compatible" with heparin in in vitro tests but more thrombogenic than a heparinized surface in a canine arteriovenous shunt.
The previously mentioned study of Muranmatsu et al., id., further confuses the issue since that study demonstrates that an increased concentration of sulfonic acid groups in a polymer leads to a higher amount of platelet adhesion. Jozefowicz et al., id. and Sorm et al., J. Polym. Sci., Polym. Symp., 66, 349 (1979) contend that carboxylic functionality is also essential for heparin-like activity. Sederel et al., J. Biomed. Mater. Res., 15, 819 (1981) synthesized a polyelectrolyte with N-sulfate and carboxylate groups that showed anticoagulant activity in several in vitro trials. Ebert et al., id., found that heparin anticoagulant activity decreased as the degree of carboxylic derivatization increased. Therefore, it appears that the carboxylate and sulfonate functionality play a role in anticoagulant action.
Jozefowicz and coworkers have examined the mechanisms involved in the function of heparinoid materials. In one study, the antithrombotic activity of crosslinked polystyrene was related to the surface density of sulfonate groups. Kanmangne et al., Biomaterials, 6, 297 (1985). Other studies involved the preparation and the properties of dextran derivatives, and demonstrated that the anticoagulant activity of these polysaccarrides was due to methylcarboxylic and sulfonated benzylamide groups. Mauzac et al., Biomaterials, 3, 221 (1984); Mauzac et al., Biomaterials, 5, 301 (1984) and Fisher et al., Biomaterials, 6, 198 (1985). Recently, these studies have recently been extended by the addition of various amino acid substituents to substituted dextran resins.
Sorm et al., id., prepared a number of synthetic polymers base on poly(methyl methacrylate) and derivatized polymers with sulfate, carboxylate and sulfamide groups in various proportions. The thrombogenicity was determined with an in vitro test of the coagulation time of plasma in the presence of thrombin. It was determined that the highest coagulation activity was found with a copolymer containing a relative amount of 86 percent (of total ionic content) sulfate groups and 14 percent carboxylate groups. The presence or absence of sulfamide groups was not found to have effect on any thrombogenicity.
Helmus et al., J. Biomed. Mater. Res., 18, 165 (1984) examined the role of surface charge of various copolymers of (L-glutamic acid co-L-leucine) and related the surface charge to thrombus formation in implanted vascular grafts in dogs. The initial ionic state controlled the biological interactions. When surface concentrations of non-ionized glutamic acid were less than 10 percent of the maximum, the amount of thrombus formed was a linear function of the degree of ionization. When 10 percent or more of the total surface sites comprised ionized glutamic acid residues, no thrombus was formed, only adhesion of single platelets to the surface was observed. The surface exposed in their canine model showed endothilization upon long blood exposure times, but that event was correlated with the extent of thrombus formation on the surfaces, with the surfaces showing the most extensive thrombus formation also showing the most endothelization.
At present, most ion-containing materials are "model ionic compounds" and do not possess adequate mechanical integrity for biomedical applications. Many of these polymers are hydrogels which must be bonded to a substrate having the necessary properties for the desired application.
As noted above, many types of heparinized polyurethane block copolymers have been tested for blood compatibility, with examples of relatively successful [Heyman et al., J. Biomed. Mater. Res., 19, 419 (1985) and Shibuta et al., J. Biomed. Mater. Res., 20, 971 (1986)]and unsuccessful [Van der Lei, id. and Lelah et al., id.] attempts being reported. Studies of "heparinoid" polyurethanes, however, are less common.
One of the first investigations of polyurethanes was by Rembaum et al., Biomat. Med. Dev. Art. Org., 1, 99 (1973). Polyether polyurethanes containing positive charges in the backbone (cationomers) were synthesized by incorporating a tertiary amine into the hard segment and reacting that group with an alkyl halide. The particular cationic polyurethanes were not studied, but they were reacted with sodium heparin to yield polyurethane-heparin complexes. A chronic carotid artery-jugular vein canine shunt was used to evaluate the thrombogenicity of this complex together with a commercial polyurethane and silicone rubber. While little difference was observed in the rates of platelet deposition on non-heparinized polyurethane or silicone rubber (although the silicone rubber was shown to cause the formation of more emboli), a retardation in platelet deposition was observed for the polyurethane-heparin complex.
Ito et al., J. Biomed. Mater. Res., 20, 1157 (1986) examined anionic polyurethanes with carboxylic acid functionality. The anionic polyurethane selectivity adsorbed albumin, did not cause a conformational change of plasma proteins adsorbed and suppressed the adherence and deformation of platelets, but did not deactivate the clotting system. Thus, the polyurethane was considered moderately thrombogenic. A heparin-bound derivative of this anionic polyurethane was not favorable for albumin adsorption, caused plasma protein denaturation and induced platelet adherence and activation, but did not activate the clotting system (as measured by thrombin times). The question of "biocompatibility" is not yet resolved for these materials, as one would presume that neither platelet activation nor activation of the clotting system would be desirable.
Two separate studies by Cooper and coworkers [Lelah et al., J. Biomed. Mater. Res., 18, 475 (1984) and Lelah et al., "Blood Compatibility of Polyethylene and Oxidized Polyethylene in a Canine Ex Vivo Shunt: Relationship to Surface Properties," in Polymers as Biomaterials, Shalaby et al (eds.) Plenum Press, New York, 257-277 (1984)] demonstrated that ionization of polyurethanes is a useful technique for improving blood compatibility.
In the first study, two uncharged polyurethanes based on 21.5 and 38 weight percent methylene bis(p-phenyl isocyanate) MDI), N-methyldiethanolamine (MDEA), and poly(tetramethyleneoxide) (PTMO) having a number average moleclar weight of about 1000 were examined using the canine ex vivo series shunt technique. Also studied were the sulfonate-containing zwitterionic, neutralized anionic, and quarterinized cationic derivatives of the MDEA-chain-extended base material containing 38 weight percent MDI.
The platelet deposition profiles of the polyurethane zwitterionomer and anionomer were more thromboresistant than the uncharged polyurethane, while the polyurethane cationomer was the most thrombogenic material of the series. The thromboresistance of the zwitterionomer correlated with a high concentration of the mobile side chain ionic sulfonate group at the surface. Ionic mobility at the interface appeared to strongly influence the blood response to these materials.
Platelet deposition profiles from a second study showed that for a non-ionized polyurethane containing 24 weight percent MDI and the analogous zwitterionomer, zwitterionization was found to improve the thromboresistance of a non-ionized material. The exact mechanisms of this action, however, were not investigated.
The base polymer utilized in the second study (in place of the MDEA-chain-extended system) was based on MDI, PTMO having a number average molecular weight of about 1000, and 1,4-butanediol (BD). The use of butanediol as a chain extender provides a base material with superior physical properties to those observed with an analogous polymer chain-extended with MDEA. This is attributed to the superior ability of the hard segments in the BD-chain extended system to aggregate and crystallize. Lelah et al., Polyurethanes in Medicine, CRC Press, Boca Raton, Fla. (1986). Butanediol is often used in commercial products, and BD is the chain extender used in PELLETHANE polyurethanes (Lelah et al., id.) and in DESERET VIALON polyurethanes.
Therefore, in spite of many prior art disclosures in the area of biocompatible materials, a need still exists for improved polymeric materials that are more suitable for blood-contacting applications, and which possess the desired bulk physical and surface properties.